Urogenital infection due to Chlamydia trachomatis (CT) is one of the most common bacterial sexually transmitted infections (STIs) and is a major public health problem worldwide. Molecular characterisation of CT is important for understanding the pathophysiological mechanisms of chlamydial disease and its transmission dynamics in sexual networks. Traditionally, strain typing of CT was based on serotyping methods characterising the major outer membrane protein (MOMP). With the advent of polymerase chain reaction and sequencing the era of molecular typing began. Molecular characterization of CT strains is based on sequence analysis of ompA gene encoding MOMP. However, in due course of time, improvements were made to enhance the discriminatory power of sequencing and quality of epidemiological information. New high-resolution genotyping methods using multiple loci such as multilocus sequence typing (MLST) and multiple loci variable number of tandem repeats (MLVA) were developed but were unable to differentiate mixed infections (MIs). The development of DNA-hybridisation methods emerged as a major breakthrough in detecting MIs. Although MLST and MLVA are more discriminative than other genotyping methods, they are laborious and expensive. DNA microarray technique is an affordable alternative for genotyping. Since recombination is widespread in the CT genome, ompA is not a reliable marker for phylogenetic studies; hence, whole genome sequencing may provide maximum phylogenetic resolution of CT strains. A descriptive review is provided of the various molecular CT typing methods. The vital information gained can be used for formulating screening programmes, targeted prevention and optimising therapeutic measures aiming to reduce disease transmission.

Chlamydia trachomatis (CT) is the most common causative agent of non-gonoccocal urethritis and has shown a worldwide prevalence of 1%–6%.[1],[2],[3] Chlamydial infections are mostly asymptomatic, leading to complications such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility in females and epididymitis and proctitis in men.[4],[5],[6],[7] These unrecognized, infected individuals act as a reservoir and transmit infections to their sexual partners. Furthermore, chlamydial infections are associated with a 3–6-fold increase in the transmission and acquisition of human immunodeficiency virus (HIV) infection.[8],[9],[10],[11]

The circular genome of CT comprises a chromosome of more than 1 million bp and a highly conserved plasmid of 7.5 kb which is present in multiple copies (7–10) within the cell.[12] Recombination and gene transfer can occur frequently in CT, the diagnostic and clinical effect of which remains largely unknown. Moreover, strains infecting different subsets of population, namely, men having sex with men (MSM), heterosexuals and bisexuals, are also genetically distinct.[13] In addition, variation in strains due to mutations may have genetic effect on pathogenicity. Hence, strain typing of CT is helpful in: (i) determining the organ or tissue tropism of strains; (ii) identification and differentiation of persistent infection, reinfection and new infection; (iii) understanding the transmission dynamics in sexual networks partner notification; (iv) evolutionary surveillance of specific clones.

In this descriptive review, we have tried to collate the available information regarding different typing methods for CT and their applications to understand the wide clinical spectrum of chlamydial disease.

~ Typing Chlamydia Trachomatis : Serotyping to Molecular Typing

Traditional strain typing of CT was based on serotyping methods such as immunofluorescence, enzyme immunoassay and radio immunoassay.[11] Serotyping is based on characterisation of the major outer membrane protein (MOMP) using specific antibodies. However, the major drawback with serotyping was that it required propagation of CT in cell cultures, which was laborious, cumbersome and less sensitive. It depended on the limited available panel of monoclonal antibodies, and hence, the detection and discrimination power were less, with the possibility of false negative or cross-reactive results.[14] The cell culture, direct detection of the bacterial antigens, and the direct immunofluorescence assay, which were until the mid-1990's the tools of direct diagnosis, have been largely replaced by the nucleic acid amplification tests (NAATs) for diagnosing or screening chlamydial infections.[15] Due to these limitations, the applications of serotyping in clinical laboratories and epidemiological studies were hampered.

With the ingenious invention of polymerase chain reaction (PCR), the era of molecular typing or genotyping began. The major molecular typing methods for CT include PCR-restriction fragment length polymorphism (RFLP), fluorescent PCR, PCR-sequencing, DNA-hybridisation, DNA-microarrays and whole genome sequencing (WGS). Molecular typing of CT is based on sequence analysis of the ompA gene encoding for MOMP. The ompA gene is 1.2 kb long and contains four highly polymorphic, variable domains (VD I–IV); each with a length of 40–100 bp [Figure 1], with VD II being the most discriminatory region. These VDs are flanked between five constant domains (CD I–V).[16] Change in the amino acid sequences in VD region accounts for the differences among CT serovars. So far, 19 different serovars of CT have been identified. Serovars A–C are commonly associated with trachoma, D–K primarily cause urogenital (UGT) infections and L1–L3 are lymphogranuloma venereum (LGV) agents, causing a more invasive disease.[17],[18]

Because of the improved turnaround time and better sensitivity, molecular typing is a great improvement and supplement for serotyping. Clinical isolates can be directly genotyped without the need for cultivation. It was these genotypic methods only, which described the genovariants such as Ja and L2b in addition to the recognised 19 serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a and L3).[19]

~ Molecular Typing Methods

Restriction fragment length polymorphism

Historically, one of the most used techniques for genotyping of CT was the ompA gene PCR-RFLP analysis. Typing of the clinical specimens by this method requires initial amplification by PCR using specific primers. The PCR-amplified ompA gene fragment can be quickly typed using restriction enzymes; Alu I for initial digestion and then digestion with another restriction enzyme (Hinf I, EcoR I, Dde I, Cfo I or BstU I) [Figure 2].[20] Different genotypes of CT produce fragments of different lengths, which can be identified by electrophoresis on agarose or polyacrylamide gels [Figure 3]. Studies have shown concordance between serotyping and ompA RFLP genotyping patterns to be around 95%.[21]

Since bacterial load in clinical specimens may be low and as the ompA gene is a single copy gene, to obtain adequate material for RFLP, a nested PCR was developed by Frost et al.[22] They reported the distribution of CT types in 46 UGT samples, with genovar E being the most prevalent type followed by genovars D and F. Later, several other studies from various parts of the world among heterosexual populations also reported a similar distribution.[23],[24],[25],[26],[27],[28] A study from India also reported genovar E to be the most predominant followed by genovars D and F in patients with infertility.[29] Another study by Rodriguez et al. also pointed out that type E was not only the most prevalent type but also very stable, with few changes even between strains from different geographical origins.[30] These findings indicate that a large proportion of infections are caused by a small number of genovars and that geographical variance exists. Various studies and the major genotypes reported worldwide using different typing methods are summarized in [Table 1].

Table 1: Comparison of different typing techniques of Chlamydia trachomatis

However, unlike heterosexuals, the predominant genovars reported in MSMs are G, D and J.[30],[31],[32],[33] Genovar G was reported for the first time from India in a bisexual male who was co-infected with Ureaplasma parvum and HIV.[34] Detection of this genotype suggests importation of new strain into the population that probably may have occurred by sexual contact with a person from a geographically distinct area.

PCR-RFLP neither requires culture of CT isolates nor the production and purification of monoclonal antibodies for CT, and hence, the technique is faster and easier to perform. Thus, it was widely applied in epidemiological studies of CT worldwide in the late 20th century and even though more modern techniques are available, RFLP is still popular in the 21st century.

However, RFLP has its own limitations such as emergence of atypical restriction patterns due to mixed genotype infections, artefacts from the enzymatic digestion and ambiguities due to polymorphisms in the restriction sites or ompA recombinants. The result of some atypical patterns requires cumbersome and time-consuming analysis and/or additional runs with different restriction enzymes. As RFLP is a single locus typing method, the discriminatory power is low, and also it does not allow for identification of single nucleotide changes, which are the most predominant variations in ompA gene.[35] Moreover, gel-to-gel variation and inter-laboratory differences make comparison of results among laboratories difficult.

~ Florescent Polymerase Chain Reaction

Use of florescent dyes improved the PCR techniques and it was possible to quantify the DNA and view the PCR reaction in real time. Fluorescent dyes were either combined with the newly amplified DNA by PCR or on specific probes for the targeted gene. The results were interpreted by detecting the fluorescent signals. Two main fluorescent PCR systems used in genotyping CT were real time-PCR (RT-PCR) and high resolution melting analysis (HRMA).

RT-PCR has been applied for genotyping CT, and using different fluorescent-labelled probes designed for ompA, CT can be genotyped by detecting different coloured fluorescences.[36] Due to distinctive fluorescences and multiple detection, channels RT-PCR can also detect mixed infections (MIs). The major drawback, however, is that due to limited number of detection channels, full genotyping cannot be completed in single assay, which makes this technique time and labour intensive.

Another method using the principle of RT-PCR is HRMA, where instead of labelling probes with fluorescent dyes, the double-stranded DNA is labelled. DNA sequence variants of CT can be identified by analysing the changes in melting curve shape and melting temperature.[37] Normalisation and comparison of these curves allow the determination of the different genotypes. Even very slight differences if present can be distinctly seen in melting curve. The performance of HRMA, however, relies on PCR conditions and concentration of initial DNA templates.[37] If the quality and amount of the starting templates is not good, it will result in untyped samples or unsuccessful typing.

~ Sequencing

ompA gene sequencing

Since the advent of sequencing, the sequence analysis of ompA gene encoding for ompA protein, has been widely used for epidemiological studies of CT. Compared with other genotyping methods, sequencing shows a higher resolution and detects even slight variations in the CT gene. Most studies have reported genotypes E, F and D as the predominant types.[2],[38],[39],[40],[41]

However, in due course of time, improvements were made to enhance the discriminative power of sequencing and the quality of epidemiological information. Therefore, genetic typing methods using multiple loci such as multi-locus sequence typing (MLST) and multi-locus variable number tandem repeats analysis (MLVA) were developed to understand the population genetic structure, diversity of species and to evaluate the association between genotype and disease.[42],[43],[44] MLST and MLVA are increasingly being used for genotyping of CT and their discriminatory indices are much higher compared with serovar or genovar typing.

Multi locus sequence typing

MLST is a typing method that uses data from several polymorphic loci, and hence, more information about the variance can be achieved. MLST is based on PCR amplification of housekeeping genes, subsequent DNA sequencing and assignment of allelic numbers according to a reference database, which in the end provides the sample with a genetic profile. The slowly evolving housekeeping genes are known to be genetically stable due to their pivotal role in cell survival, and therefore, MLST is a suitable tool for evolutionary studies.

The first MLST system for CT was given by Klint et al., in 2007.[42] Through a computational approach, the highly conserved genome of CT was analysed, not for housekeeping genes but for highly variable genetic regions. The MLST of CT was done by sequence determination of the five genomic targets in the genome plus ompA. These five regions consist of three hypothetical genes (CT058, CT144, CT172) and two known genes (hctB and pbpB).[42] The variations in these regions mainly consist of point mutations. The hctB gene encodes a histone H1-like protein, which functions to regulate chromatin structure, gene expression and transition of reticulate body to elementary body. The highly variable pbpB gene encodes a penicillin binding protein. This MLST system has been found to be significantly more discriminatory than ordinary ompA genotyping.[45] On an analysis of 47 clinical isolates by MLST system, Klint et al. were able to determine 32 genetic variants while ompA determined only 12 variants.[42]

Since 2007, two more MLST systems for genotying of Chlamydiciae bacteria have been published.[43],[46] Bom et al. improved the technique using nested PCR before sequencing thus increasing the sensitivity.[42],[46] This helped in typing even those clinical samples having low bacterial load. An alternative MLST system developed by Pannekoek et al. was used for analysing evolutionary changes and was based on sequencing seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hflX, oppA-3) plus ompA gene.[43]

Multiple loci variable number of tandem repeat analysis

The discriminatory power of CT typing is increased when information on multiple parts of the genome and plasmid is obtained. MLVA is one such method and is based on PCR amplification and DNA sequencing of ompA and several variable number of tandem repeats (VNTRs) at different locations.[44] VNTRs are defined as repeated stretches of the same nucleotide or motifs. Several of these genomic VNTR loci are used in MLVA and the extent of repetition results in a sequence type (ST), that is, used for cluster analysis.[47] It is known that in these areas of repetition, DNA polymerase is prone to error during replication (especially in single nucleotide repeats), so this type of target is well suited for short term, local epidemiological studies. Analysis of several VNTR targets results in high-resolution power.

The method was first proposed by Pedersen et al., in 2008[44] where they selected three loci (CT1335, CT1299 and CT1291) with VNTR along with ompA for PCR amplification and DNA sequencing, and in the subsequent studies, up to eight loci were used.[48] The MLVA system was shown to have a comparable resolution to the MLST system.

Various studies have revealed that ompA genotyping is far less discriminatory compared to high resolution MLST and MLVA.[13],[45],[49],[50],[51],[52],[53],[54] The Simpson's index of diversity [55] or discriminatory index (DI) for both MLST and MLVA is quite high and fulfils the recommended value of ≥0.95, as per the consensus guidelines by the European Society of Clinical Microbiology and Infectious Diseases Study Group on Epidemiological Markers.[56] A study by Gravningen et al. showed that MLST provided 4.5 times higher resolution in comparison to ompA gene sequencing and reported a total of 50 STs from 248 clinical isolates.[13] A study showed that MLST and MLVA if used together have a DI almost equal to 0.99.[47] However, sequencing including MLST and MLVA has its own limitations. A comparative analysis of different typing methods for CT is shown in [Table 2]. Sequencing is incapable to differentiate MIs and can cause superimposed peaks in the chromatograms. This is one of the important reasons of untyped or non-interpretable results. Various other factors, namely, impure/inadequate concentration of PCR products, failure of conjugation formation between PCR products and sequencing primers can also impact the sequencing results and the failure ratio can be sometimes even more than 10%. Moreover, MLST is limited by the fact that only fraction of the genome is used for typing and so samples that are indistinguishable with respect to the target regions may still have considerable variation in the remaining DNA.[59]

Table 2: A summary of various studies on molecular typing of Chlamydia trachomatis

Although sequencing resolved the problem of resolution for typing CT yet it was unable to differentiate MIs. Hybridisation methods for genotyping emerged as a major breakthrough in detecting MIs and have been used in various epidemiological studies.[57],[58],[62] The advanced hybridisation systems being used are reverse line blot hybridisation (RLB-h), reverse dot blot hybridization (RDB-h) and microsphere suspension array hybridisation (MSA-h).

Reverse line blot hybridisation

The method was introduced in the beginning of 21st century.[63] The amplified DNA of CT is hybridised with oligonucleotide probes labelled on nylon membrane with amine. Thereafter, the DNA is marked with streptavidin peroxidase, which later reacts with chemiluminescence detection reagents, and hence, at the end of the reaction, there will be a blot on the exposed detection film.[63] On a labelled membrane, up to 45 lanes are available for specimens in a single assay and each lane can be labelled with a maximum of 43 probes at a time.

Using various genus and species specific probes, RLB-h can detect multiple CT infections [60] which are otherwise expressed as non-interpretable by sequencing. Various epidemiological studies performed using RLB-h demonstrated that MIs due to CT could range from 8% to 25% among the various study populations.[57],[58],[62] In a study from Australia,[57] multiplex PCR-RLB-h and nested PCR-RLB-h were used simultaneously to type 191 CT isolates. The results showed 166 single infections and 25 MIs including 3 cryptic plasmid deficient samples. The probe labelled membranes used in RLB-h are reusable and the method can also be used for typing other organisms, cell alleles and polymorphisms which makes RLB-h an economic and effective genotyping method.[64] The major limitation with RLB-h is the high turnaround time of about 1.5 days.

Reverse dot blot hybridisation

Principle of RDB-h is somewhat similar to RLB-h; however, RDB-h is faster and easier to perform than RLB-h. It does not require a machine and related exposing reagents to blot and the results can be read optically.[59] Also, RDB is more flexible for sample arrangement as each membrane is an individual disposal for one sample and the number of membranes is decided according to the number of samples to be tested. However, in RLB-h, a miniblotter with 45 lines needs to be prepared irrespective of the number of samples.

Microsphere suspension array hybridisation

MSA-h is also a genotyping method based on hybridisation and instead of coating probes or sample DNA to a membrane (as in RLB-h and RDB-h), in MSA-h, the 5'-end amino C-12 modified probes are coated to carboxylated beads (microspheres) covalently. Amplified DNA hybridised with probes is incubated with streptavidin-R-phycoerythrin to get dyed and the results are interpreted by detecting and analysing the fluorescent signal which are expressed as median fluorescent intensity value.[65] As different type of probes can be used in the same liquid system, MSA-h is suitable for multiple PCR simultaneously. Moreover, the sensitivity of MSA-h is highly improved as microspheres have numerous locations for probes to label. The technique is faster, easier to understand, has an advantage of high multiplexing capability and has an automatic software-based result interpretation system.[65] Various studies have proved MSA-h as a high throughput method suitable for identifying multiple infections. Using MSA-h, a study from China showed that 18.3% of the population had multiple infections with serotypes J and K.[60]

The major drawback with hybridisation methods is the resolution, which is relatively lower than sequencing. As these methods depend on the fixed probes, the novel genovariants can hardly be identified with these techniques.

~ Dna Microarray

Although MLVA and MLST are more discriminative than other major genotyping methods, they are expensive and equipment dependent, and hence, their implementation in routine diagnostic laboratory is difficult. Diagnostic DNA microarray technology has recently emerged as a promising alternative in microbial genotyping. The technology helps avoid extreme sequencing and has been used in research as well as routine diagnostic laboratories.[35],[61] Based on the target regions of the MLST system, a multilocus typing DNA (MLT-DNA) microarray was developed by Christerson et al.,[66] in 2011. The method was less expensive, faster and had a sensitivity comparable to MLST.[66] The results showed that MLT-DNA microarray had high resolution (2.4 times higher than ompA sequencing) with 100% specificity and high throughput of 96 samples in parallel. The analysis can be performed within a single working day, which is far less in comparison to MLST analysis (3–4 days). Furthermore, the cost of equipment and the consumables required for MLT-DNA microarray are considerably cheaper than required for DNA sequencing. Being software based (MLT Line), the data processing and genotype assignment are easily done. On the contrary, DNA sequencing requires through data analysis, which requires extensive expertise of laboratory staff. MLT-DNA microarray can be used in studies where rapid genotyping of CT is needed to achieve the crucial epidemiological information.

~ Whole Genome Sequencing: the Futuristic Approach

WGS is necessary to understand the evolution, diversity and epidemiology of CT. It may, therefore, provide maximum phylogenetic resolution, in-depth understanding of the population structure and the patterns of CT infection. Studies have confirmed that ompA is not a reliable marker for phylogenetic studies due to its extensive recombination within the genovars of CT strains.[67],[68] Use of full genome sequence data in hospital settings has been demonstrated in recent studies to better understand the epidemiology of CT.[69]

Until recently, cell culture has been necessary to generate sufficient CT DNA for WGS.[12] This has been a major technical hurdle in the production of pure genomic DNA, which in turn made large-scale comparative genomic studies difficult. The problem was solved by introduction of culture-independent techniques for generating sufficient amount of DNA (directly from clinical samples) required to determine WGSs of CT.[12],[70] One of these methods involved the use of MOMP binding antibodies attached to magnetic beads to pull down CT-infected cells from the clinical samples.[71] This technique could not be used for samples collected in devices for commercial assays that contains a lysis buffer which disrupts the MOMP structure and hence presents the antibody binding. Another cell culture-independent method used the microdroplet PCR introduced by Raindance technologies (Lexington, MA, USA) to enrich the chlamydial sequences in the crude diagnostic DNA extracts.[70] After next generation sequencing, CT genomes were reconstituted and compared for single nucleotide polymorphisms (SNPs) using the existing known sequences to identify the single or mixed CT strains directly in the clinical samples.

WGS of different CT strains elaborated the data on the similarities and discordances of various genomes with different genovar (ompA) types. A study by Harris et al.[68] demonstrated that exchange of the whole or part of the ompA gene is a natural phenomenon in distinct lineages of CT. They suggested that early in the evolutionary history CT has split into two distinct clades representing the UGT and the LGV biovars. UGT clade is comprised two lineages (T1 and T2) separated from their common ancestors by 2374 and 2228 SNPs, respectively. T1 lineage is composed of clinically prevalent UGT serotypes, whereas T2 lineage contains the rarer UGT serotypes. All ocular strains form a cluster within the T2 lineage and indicate that they emerged from a UGT ancestor. They also found lack of variation within the L2b genovars and suggested it as indicative of rapid transmission and the emergence of L2b epidemic due to clonal outbreak.

~ Molecular Typing: Applied Aspects

Monitoring sexual networks

Molecular typing methods have been used to elucidate various clinical, evolutionary and epidemiological questions. Before the advent of molecular typing methods, the evaluation of STI transmission dynamics was mainly performed using source and contact tracing. This was laborious and also biased by the information provided by the index patient and the approach and attitude of the interviewer toward the patient. With the high-resolution typing methods, sexual networks can be unveiled with a lot more precision and less bias.

Recurrent STIs including CT are quite common among the high risk groups (CSW, MSM) and young adults because of the ongoing exposure. Careful and proper interpretation of repeated detection of CT even after the treatment is important for monitoring patient management, partner notification and treatment evaluation. Redetection after therapy probably indicates, (i) a new infection by another (new) infected partner; (ii) re-infection due to re-exposure to a partner not identified or inadequately managed through partner notification and; (iii) persistent infection due to treatment failure. Hence, typing and comparing CT strains detected before and after treatment can identify the cause of redetection. Discordant strain types explain a new infection while the concordant types explain probably a re-infection or a persistent infection.

Increasing numbers of LGV cases were reported over recent years in Europe, United Kingdom, North America, Australia, etc.[72] Majority of these infections were ano-rectal, and amongst the HIV co-infected MSMs.[16],[72],[73] The ompA gene sequencing of the LGV positive samples from Amsterdam revealed a new CT genovariant, L2b.[16] Recently, MLST analysis identified the L2b strain circulating preferentially among specific subpopulations within the MSM community in Amsterdam, suggesting the role of network assisted factors such as sexual host behaviour and bacterial factors like tissue tropism in its emergence.[46] Recently, two new hyper virulent LGV strains viz., L2c and L2/L2f have also been identified.[74] Although the LGV genovars do not have any additional genes that account for more invasive and chronic infections as compared to uro-genital genovars, many genes in the circular genome and the plasmid of LGV genovars differ in their sequences. This probably may have some role in the varied clinical presentation and disease outcome due to LGV genovars and genotyping of LGV strains may further elucidate about it.

The clinical presentation, patient management and treatment modalities are different for infections due to LGV genovars. Hence, it is quite imperative to differentiate patients infected with LGV genovars from those infected with non-LGV genovars. LGV is more commonly seen among MSM co-infected with HIV and the disease presentation is more destructive, sometimes requiring additional clinical management and follow up.[75] As compared to the non-LGV strains, prolonged treatment is required for anorectal infections caused by LGV strains; 1 week of doxycycline versus 3 weeks, respectively.[76]

Studies have shown that a significant proportion of patients with anorectal LGV infections are asymptomatic on presentation.[71] Hence, LGV typing of CT positive anorectal samples is important in symptomatic and asymptomatic patients. It is recommended to screen all MSM who report receptive anal sex during last 6 months, for anorectal CT infection and further type the positive rectal samples for LGV genovars.[77]

Chlamydia trachomatis and emerging mutations

There was a sudden and unexplained dip in the number of reported CT infections from Sweden in 2006. Later a new strain called 'new variant CT' (nvCT) was found responsible for this diagnostic resistance. The nvCT was characterised by a 377 bp deletion in the p LGV 440 gene of a cryptic plasmid which is the target area for some of the NAATs [78] and hence these tests failed to detect nvCT infections. Although the emergence of nvCT was the first reported example of mutation in CT and the strain remained confined to Sweden and Norway, still it highlights the importance of CT sequence typing to monitor the emerging mutations that circulate in the population. Later adjustments in diagnostic tests were made and in some cases multiple genetic regions for target detection were introduced to accurately detect this strain. The strain was later recognised as a Serotype E and further typing using new systems has strongly suggested the spread of a single clone.[79]

Utility of molecular typing in cases of child sexual abuse

The presence of STIs including CT in a child is often used to support the allegations of sexual abuse (SA) and in some cases, may prompt an investigation of possible abuse. Postnatally acquired CT is usually considered as diagnostic indicator of SA and its identification beyond the perinatal period almost always suggests SA.[80] However, this may not hold true always as detection of CT in UGT samples from young children is not always a consequence of conventional sexual contact.

Autoinoculation of CT from the ocular infection to the UGT site is a well-established phenomenon and leads to the detection of CT in UGT specimens even in the absence of any sexual contact.[81] This particularly is relevant in a country like India, where trachoma still remains endemic and is not considered as an STI. The relevance of genotyping in such situation cannot be underscored and it is important to include CT genotyping into formal guidelines for examining the source of STIs in young children in areas where trachoma genotypes may continue to circulate. Also, it is important to know the trachoma and UGT genotypes circulating in the conventional adult sexual networks in the area, so as to establish the possible route of transmission.

~ Conclusion

To conclude, the high prevalence of CT infections reinforces the importance of routine screening as well as molecular typing, to decrease the burden of chlamydial infections. Molecular typing of CT has helped in elucidating the wide clinical spectrum of chlamydial disease and is a useful tool in epidemiological studies, investigation of infection transmission, surveillance of emerging genotypes or genovariants in populations and understanding the genetic causes behind varied pathophysiological mechanisms of disease. Molecular epidemiological studies provide insight into the transmission dynamics of CT infections and could therefore help to detect specific populations and networks at risk. The vital information gained can be utilised in the formulation of screening programs, targeted prevention and optimizing therapeutic measures, aiming to reduce disease transmission.

Lister NA, Smith A, Read T, Fairley CK. Testing men who have sex with men for Neisseria gonorrhoeae and Chlamydia trachomatis prior to the introduction of guidelines at an STD clinic in Melbourne. Sex Health 2004;1:47-50.

Qin X, Zheng H, Xue Y, Ren X, Yang B, Huang J, et al. Prevalence of Chlamydia trachomatis genotypes in men who have sex with men and men who have sex with women using multilocus VNTR Analysis-ompA typing in Guangzhou, China. PLoS One 2016;11:e0159658.

de Vrieze NH, van Rooijen M, Schim van der Loeff MF, de Vries HJ. Anorectal and inguinal lymphogranuloma venereum among men who have sex with men in Amsterdam, the Netherlands: Trends over time, symptomatology and concurrent infections. Sex Transm Infect 2013;89:548-52.

de Vries HJ, Zingoni A, Kreuter A, Moi H, White JA; European Branch of the International Union against Sexually Transmitted Infections; European Academy of Dermatology and Venereology; European Dermatology Forum; European Society of Clinical Microbiology and Infectious Diseases; Union of European Medical Specialists; European Centre for Disease Prevention and Control; European Office of the World Health Organisation 2013. European guideline on the management of lymphogranuloma venereum. J Eur Acad Dermatol Venereol 2015;29:1-6.